Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media

Embryo metabolism is an indicator of viability and, therefore, efficiency of the culture medium. Currently, little is known regarding porcine embryo metabolism. The objective of our study was to evaluate glucose and pyruvate uptake and lactate production in porcine embryos cultured in two different media systems. Oocytes were matured and fertilized according to standard protocols. Embryos were allocated randomly into two culture treatments, NCSU23 medium or G1.2/G2.2 sequential culture media 6-8 h post-insemination (hpi). Embryo substrate utilization was measured at the two-cell (24-30 hpi), 8-cell (80 hpi), morula (120 hpi), and blastocyst (144 hpi) stages using ultramicrofluorimetry. Glucose uptake was higher (P < 0.05) in two-cell embryos cultured in G1.2 than in NCSU23 medium (4.54 +/- 0.71, 2.16 +/- 0.87 pmol/embryo/h, respectively). Embryos cultured in G1.2/G2.2 produced significantly more lactate than those in NCSU23 at the eight-cell stage (9.41 +/- 0.71, 4.42 +/- 0.95 pmol/embryo/hr, respectively) as well as the morula stage (11.03 +/- 2.31, 6.29 +/- 0.77 pmol/embryo/hr, respectively). Pyruvate uptake was higher (P < 0.05) in morula cultured in G1.2/G2.2 versus NCSU23 (22.59 +/- 3.92, 11.29 +/- 1.57 pmol/embryo/h, respectively). Lactate production was greater (P < 0.05) in blastocysts cultured in G1.2/G2.2 (38.13 +/- 15.94 pmol/embryo/h) than blastocysts cultured in NCSU23 (8.46 +/- 2.38 pmol/embryo/h). Pyruvate uptake was also greater in blastocysts cultured in G1.2/G2.2 (24.3 +/- 11.04) than those in NCSU23 (11.30 +/- 2.70). When cultured in NCSU23 medium, two- and eight-cell embryos utilized less glucose than morulae and blastocysts, and two-cell embryos produced less lactate than blastocysts (P < 0.05). In G1.2/G2.2 media, two-cells took up less pyruvate than morulae or blastocysts, while blastocysts produced more lactate and utilized more glucose than two-cell, eight-cell and morula stage embryos (P < 0.05). As in other species, glycolysis appears to be the primary metabolic pathway in post-compaction stage porcine embryos. Culture medium composition affects not only substrate uptake, but also metabolic pathways by which these substrates are utilized in porcine embryos at several developmental stages.     

Development of sheep preimplantation embryos in media supplemented with glucose and acetate

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF) + sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.     

Development and viability of in vitro derived porcine blastocysts cultured in NCSU23 and G1.2/G2.2 sequential medium

Porcine embryo development in vitro is relatively inefficient compared to other domestic species. Currently, a single culture medium (NCSU23) is the standard for porcine in vitro systems. However, the G1.2/G2.2 sequential culture system has been beneficial for embryo development in other species. The objective of this study was to compare porcine preimplantation embryo development in vitro and subsequent blastocyst viability and metabolic activity using NCSU23 and G1.2/G2.2 culture media. Oocytes were matured in defined TCM199 base medium for 45 to 47 h and fertilized in mTBM for 4 h. Embryos were cultured in either NCSU23 for 146 h or G1.2 medium for 72 h followed by culture in G2.2 medium for an additional 74 h. Blastocyst substrate use was measured using a modification of the hanging drop technique. Culture in NCSU23 resulted in a higher percentage (P < 0.05) of embryo cleavage (74.0%) and blastocyst development (14.6%) than culture in G1.2/G2.2 (67.8% and 7.8%, respectively). Both NCSU23 and G1.2/G2.2 produced blastocysts with similar mean cell numbers (51.5 +/- 4.3 and 47.1 +/- 4.3, respectively), similar glucose use (10.81 +/- 1.39 and 10.12 +/- 1.72 pmol/embryo/3 h, respectively) and pyruvate use (1.08 +/- 0.056 and 0.88 +/- 0.048 pmol/embryo/3 h, respectively). These data indicate that a sequential culture system can support porcine embryo development in vitro without compromising embryo viability. However, the G1.2/G2.2 system was not as effective as NCSU23 in supporting blastocyst development. Sequential media should be formulated specifically for porcine embryos to improve embryonic cleavage and blastocyst development.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Morphological differences in human zygotes and embryos cultured in different media

Summary Purpose: To compare the effects of four culture media on the quality of human zygotes and embryos. Methods: Prospective study analyzing 2289 human embryos cultivated simultaneously in two different culture media: HTF, the default medium, with either Universal IVF, Global or IVF-30 as the secondary media. The sibling oocytes by each patient were randomly divided between the two culture media following intracytoplasmic sperm injection (ICSI). On day 1 the pronuclear stage of zygotes were evaluated and on day 2 embryos were evaluated according to the number of cells, percentage of fragmentation and number of nuclei. Z-test and odds ratios were used in the statistical analysis. Results: There was a higher percentage (55.2%) of class A1 + A2 zygotes with IVF-30 compared with HTF, Global or Universal IVF media (49.1%, 44.7% and 44.2%, respectively). The percentage of Top embryos was significantly higher with Global (40.2%) compared with HTF (21.3%), IVF-30 (25.0%) or Universal IVF media (11.2%). Conclusions: Global medium produced more Top embryos evaluated on day 2 of development.     

Development of in vitro matured and fertilized bovine embryos cultured in media containing human leukemia inhibitory factor

Three experiments were conducted to evaluate the effect of addition of human leukemia inhibitory factor (hLIF) to synthetic oviduct fluid medium (SOFM) supplemented with human serum (HS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) on the development of bovine embryos matured and fertilized in vitro. In vitro matured and fertilized bovine oocytes were cultured in SOFM supplemented with 10% HS to obtain embryos at 1 - cell, 4 - or 8 - cell, and morula or early blastocyst stages. In Experiment 1, embryos at the different developmental stages were cultured in SOFM supplemented with 10% HS and 1 of 6 different dosages (0, 500, 1000, 2000, 4000, 6000 U/ml) of hLIF. In Experiments 2 and 3, the embryos were cultured in SOFM + BSA and SOFM + PVA, respectively with or without hLIF (5000 U/ml). In, Experiment 1, the addition of any hLIF dosages did not improve development to the expanding blastocysts as compared with the control (without hLIF) in each embryonic stage. Embryonic stages at the time of hLIF addition affected the development; early blastocysts resulted in significantly (P<0.01) better development than the other stages. The addition of hLIF at 1 -, 4 - and 8 - cell stages in Experiment 2 and 3 had no effect on development to the expanding blastocyst stages significantly (P<0.01) improved the development. The results indicate that the effect of hLIF addition is critical to embryonic stages and the advantage of hLIF addition is only observed when SOFM is supplemented with BSA or PVA. A stimulating effect of hLIF was not observed when SOFM was supplemented with HS.     

Development retardation in cultured preimplantation rabbit embryos

Day 3 to Day 5 preimplantation rabbit embryos were cultured for 24 h in chemically defined media which are widely used in early embryo culture (BSM II and Ham's F-10) supplemented with BSA or homologous serum. For the next 24 h, the embryos were left in the same culture medium, placed in freshly made medium, or cultured in medium which was supplemented with uterine flushings. In addition, 24-h cultured embryos were transferred into uteri of synchronous recipients for 1 day. After culture or transfer, development was assessed by cell proliferation evaluated by incorporation of tritiated thymidine. In comparison to non-cultured controls, thymidine incorporation demonstrated a considerably impaired cell proliferation after culture in defined media irrespective of medium, supplement, or replenishment with fresh medium. For Day 3 embryos, there was a developmental retardation amounting to about 1 day after 2 days in culture. Compared to Day 3 embryos, delay was clearly more pronounced in Day 4 and Day 5 blastocysts, i.e. in stages which had been retrieved from the uterus before culture. Supplementation with uterine flushings markedly promoted blastocyst cell proliferation (P less than 0.001). Incorporation data examined after transfer showed that impairment of cell proliferation caused by 1 day in culture had been compensated for to a large extent within 1 day in utero.     

Development of mouse embryos co-cultured with polarized or non-polarized uterine epithelial cells using sequential culture media

This study investigated the effects of the in vitro co-culture of mouse embryos with non-polarized or polarized uterine epithelial cells, using sequential culture media, on their development to blastocysts, blastocyst quality (blastocyst diameter and cell number), apoptosis, Bcl-2 and Bax gene expression. There were three treatments, all of which used sequential culture media. The treatments were no co-culture (control), non-polarized or polarized epithelial cell monolayer co-culture in 24-well tissue culture plates. Mouse uterine epithelial cells were isolated enzymatically and were seeded either on the surface of the culture plate (non-polarized monolayer) or on a Millipore filter insert coated with extra-cellular matrix extract (polarized monolayer) that was then placed in the culture plate. Two-cell mouse embryos were cultured in G-1 ver3 medium to the eight-cell stage when they were randomly assigned to the treatments. The culture medium was G-2 ver3 during the treatment phase of the study. Significances of differences were evaluated by the one-way analysis of variance for continuous data. The epithelial cells cultured on Millipore filters became polarized and their morphology compared favorably with those cultured on the surface of the culture plate and in vivo uterine epithelial cells. After 96 h on the treatments, the polarized monolayer had supported the development of significantly more hatched blastocysts (80.0%; P<0.05) than the non-polarized monolayer (63.4%) or the control (61.4%) culture treatments. Co-culture resulted in the production of blastocysts with significantly more cells (non-polarized monolayer 56.7+/-2.1, polarized monolayer 61.9+/-2.1) than the control culture (42.8+/-2.6; P<0.05) but the diameter and shape of the blastocysts were not significantly different. The proportion of blastocysts with apoptotic blastomere was higher for the control culture (94.4%) than for the non-polarized (68.2%) or polarized (66.7%) co-culture systems (P<0.05). Moreover, the apoptotic index was significantly higher in control blastocysts (5.6+/-0.9; P<0.05) than in non-polarized (1.7+/-0.3) or polarized (1.5+/-0.3) co-culture. In the control, Bax mRNA was strongly expressed when compared to co-culture treatments (P<0.05), whereas, the relative abundance of Bcl-2 mRNA to the beta-tubulin was lower than co-culture treatments (P<0.05). It is concluded that a co-culture system involving polarized uterine epithelial cells and sequential culture media is a promising method of producing mouse embryos.     

Development of mouse embryos in media containing lectins

Lectins known to stimulate mitosis in cultured cells were evaluated for effects on development of mouse embryos in vitro. Two-cell mouse embryos were cultured in one of the following treatments: Whitten's medium as the control medium; Whitten's medium with 1, 10 or 100 mug/ml concanavalin A; Whitten's medium with 1, 10 or 100 mug/ml leucoagglutinin; Whitten's medium with 1, 10 or 100 mug/ml phytohemagglutinin; Whitten's medium with 1, 10 or 100 mug/ml pokeweed-mitogen; and Whitten's medium with 1, 10 or 100 mug/ml wheat germ agglutinin. Development to the morula stage was blocked in media with 100 mug/ml concanavalin A and 10 and 100 mug/ml wheat germ agglutinin, whereas blastocyst formation was blocked in all pokeweed-mitogen supplemented media. Embryos incubated in 10 and 100 mug/ml wheat germ agglutinin underwent premature cavitation or vacuolation at 24 to 48 h of culture. More embryos formed blastocysts in media with 1 and 100 mug/ml phytohemagglutinin and 10 mug/ml leucoagglutinin than in Whitten's medium (P<0.05). The percentage of embryos hatching was greatest in 1 mug/ml phytohemagglutinin (P<0.05), but it was the same in Whitten's medium, 1 mug/ml concanavalin A and 1 mug/ml leucoagglutinin (P>0.05). Cell division was not stimulated by the lectins; however, it was significantly suppressed in media with 10 and 100 mug/ml concanavalin A, 100 mug/ml phytohemagglutinin, 1, 10 and 100 mug/ml pokeweed-mitogen, and 10 and 100 mug/ml wheat germ agglutinin. Solubility of the zona pellucida in sodium isothicyanate (NaSCN) was reduced in 100 mug/ml phytohemagglutinin, 100 mug/ml leucoagglutinin and 1 mug/ml wheat germ agglutinin media (P<0.05) when compared to Whitten's medium and may have accounted for the reduced hatching observed in these treatments. Development of isolated blastomeres into blastocysts was reduced in media with 1 mug/ml wheat germ agglutinin, 1 mug/ml concanavalin A, and 10 and 100 mug/ml leucoagglutinin (P<0.05) but was similar in media with 1 mug/ml leucoagglutinin and 1, 10 and 100 mug/ml phytohemagglutinin when compared to Whitten's medium (P>0.05). The extent of embryo development in media with lectins depended upon the degree of cytotoxicity and potential biochemical modifications induced in the zona pellucida. Greatest embryo development took place in medium with 1 mug/ml phytohemagglutinin; however, the mechanism was not that of stimulation of cell division or a change in zona pellucida solubility.     

Cell allocation in bovine embryos cultured in two media under two oxygen concentrations

Blastocyst development, total cell number and allocation to inner cell mass (ICM) and trophectoderm (TE) lineages was compared among day 9 hatched blastocysts from four culture treatments in a two-factor design. Two modified commercial media (KSOM and SOF) were used in atmospheres with two oxygen concentrations (5% and 20% O2). No significant effect of medium on development was found, but 20% O2 increased hatching (p < 0.05). There were more cells in hatched blastocysts cultured in KSOM than in SOF (181 vs 136, respectively; p < 0.0001); however, ICM/total cell ratio was not affected by medium. There was a trend suggesting that the proportion of cells allocated to ICM was lower in hatched blastocysts cultured under 5% O2 compared with 20% O2 (0.323 vs 0.380, respectively; p < 0.1). No significant interactions between medium type and oxygen concentration were found. These results indicate that culture components used in this study may affect cell proliferation without altering cell allocation, and that oxygen concentration may play a role in allocation of cells to ICM and TE lineages.     

Donor and recipient ewe factors affecting in vitro development and post-transfer survival of cultured sheep embryos

Donor and recipient factors were assessed during development of embryos following superovulation, collection at the pronuclear and two-cell stage, culture in Synthetic Oviduct Fluid medium for 5 days and twin transfer into synchronised recipients to elucidate what factors affect embryo development and post-transfer survival. In particular, the administration of exogenous progesterone to recipients using an intravaginal CIDR^TM device immediately following embryo transfer was investigated.

From 138 embryos collected from 30 donor ewes, 75% (103) were of transferable quality following culture, of which 100 were transferred to 50 recipients. There was significant variation (P < 0.001) in embryo development to the blastocyst stage between different donor ewes, but this was not related to the donor ovulation rate. At ultrasound sonography (approximately Day 60 of pregnancy), 58% of recipients were pregnant and 42% embryos had survived. Donor ovulation rate was related to embryo survival (P < 0.05) after transfer; the survival rate of embryos from ewes with high ovulation rates was lower than that of embryos from ewes with low ovulation rates. Exogenous progesterone supplementation following transfer did not affect embryo survival, rate of embryo development or plasma progesterone levels. In general, the results from this study suggest that factors other than efficacy of embryo culture can affect the outcome of embryo survival following transfer and that, where possible, these factors should be considered and balanced in experimental designs.     

Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media.

Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.     

Implantation potential and fetal viability of mouse embryos cultured in media supplemented with platelet-activating factor

Supplementation of culture media with PAF failed to increase the proportion of 2-cell mouse embryos which developed through to the expanded blastocyst stage after 72 h of culture in vitro. Culture of embryos with 1.86 microM-PAF, however, significantly increased the number of cells in expanded blastocysts. Culture in vitro with 0.186 or 1.86 microM-PAF significantly increased the ability of embryos to implant upon transfer to pseudopregnant recipients. The specificity of this effect was demonstrated by its inhibition by the specific PAF-receptor antagonist, SRI 63-441. Of the embryos which implanted, there was no difference in the proportions of PAF-treated or control embryos which resorbed nor was there any effect of PAF on fetal weight or crown-rump length measurements. Embryos cultured with 1.86 microM-PAF did have slightly heavier placentae than their respective controls. These results demonstrate the ability of exogenous PAF to enhance the viability of preimplantation mouse embryos cultured in vitro and support the hypothesis that a major site of the action of PAF during early pregnancy is the embryo itself.     

Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media

The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F-10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared. The uterine flushings derived from donors of 0.5-6 years of age. Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration. Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation). The growth retardation was more pronounced in blastocysts (recovered at day 4 post coitum [p.c.]) than in morulae (recovered at day 3 p.c.). Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development. Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages. Advanced donor ages, heating up to 80 degrees C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos. The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium.     

Use of G1.2/G2.2 media for commercial bovine embryo culture: equivalent development and pregnancy rates compared to co-culture

The expanded application of commercial bovine IVM, IVF, and IVC systems is dependent on the ability to produce embryos in culture that are capable of producing normal pregnancies. Because serum containing culture systems can induce neonatal and fetal problems there exists a definite need for a serum-free culture system that produces viable blastocysts. This study demonstrated that the physiological sequential media system G1.2/G2.2 could produce bovine blastocysts at rates equivalent to co-culture. Additionally, these blastocysts had equivalent or increased cell numbers and inner cell mass development. Blastocysts produced in the G1.2/G2.2 culture system produced pregnancies following both fresh transfer and cryopreservation at equivalent rates to co-culture. Finally, this study demonstrated that the media system G1.2/G2.2 could be used in a commercial OPU transfer program without any loss in the numbers of blastocysts produced or the numbers of pregnancies resulting following transfer from either fresh or cryopreserved blastocysts.     

Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo

Embryos from superovulated female mice that developed in vitro from the two-cell stage were compared with in vivo embryos with respect to yield of blastocytes, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocytes in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA-dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A).oligo(dT)-dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.